The over arching hypothesis is that the brain is an important reservoir for retroviruses because of their ability to infect long lived terminally differentiated cells and viral products released from these cells can cause immune activation and neuronal injury Aim 1: To understand the mechanism of viral persistence in brain If there is any hope to eradicate HIV, close attention to the viral reservoirs in the brain is necessary. The brain is a unique site of viral latency since it infects resident macrophages/microglia and astrocytes. These cells have very low turnover rate, and the mechanism of viral entry and persistence is very different than that of lymphocytes which are the major cell type infected by the virus in the lymphoid organs. Our laboratory has focused its efforts on studying the mechanism of viral infection of astrocytes. We have found that while free viral particles can enter these cells, cell to cell contact with lymphocytes is the most efficient way to infect astrocytes. We have discovered that the virus enters astrocytes by using CXCR4 and is aided by formation of tight junctions between the cells which we have termed, viral synapses. Another interesting observation made in our laboratory is that astrocytes express CD4 on the cell surface when eposed to cytokines which makes them vulnerable to HIV infection. This helps explain why astrocytes in vivo are infected in large numbers yet these cells have been very difficult to infect in vitro with cell free virus. We have also found that upon entry, the virus can enter the endolysosomal pathway which acts as a host defense mechanism. Hence strategies than modulate these pathways could have a significant effect on the establishment of a reservoir in the brain. We are now confirming these findings using virus and lymphocytes from CSF of HIV infected individuals to determine if there are starins of HIV that preferentially infect astrocytes. In a cohort of HIV patents who have been well characterized at NIH we have found that the virus in the cells and the spinal fluid is latent when the retroviral therapy is used. We are investigating the regulatory pathways involved in maintaining the latency of the virus in the CNS. Aim 2: To investigate the mechanism of neuronal injury by HIV and endogenous retroviruses. Despite the use of antiretroviral agents and excellent control of the virus in the periphery, HIV infected patients continue to develop cognitive impairment. Currently available antiretroviral agents have no effect on the production of early viral proteins once the virus has integrated into the chromosome. One of these proteins, Tat, has been shown to be neurotoxic. Our laboratory was one of the first to demonstrate its toxic potential and has extensively investigated the mechanism by which it causes neurotoxicity. We have developed an assay quantify Tat in spinal fluid and are developing therapeutics to block the effect of Tat on HIV replication and neurotoxicity. We have recently shown that the envelope protein of an endogenous retrovirus can also cause toxicity and have implicated it in the pathophysiology of motor neuron diseases. We are studying the mechanism underlying the toxicity and developing drugs to block it. Aim 3: To develop therapeutic approaches to prevent viral activation and formation of viral reservoirs in the brain. We are generating cell lines with inducible expression of HIV-Tat protein and the HERV-K virus. These cell lines will be used in high through put screening assays to screen for anti-sense molecules and for small pharmacological compounds that suppress their production. Currently viral constructs are being prepared, which will be cloned into appropriate vectors for transfection of cell lines. In summary, we have shown that astrocytes in the brain are an important reservoir for HIV and that cell to cell contact with lymphocytes is necessary for viral entry and the lysosomal pathway in these cells regulates the intracellular trafficking of the virus and its ultimate ability to successfully infect these cells. Further, we have shown that the HIV protein Tat and the env protein of endogenous retrovirus-K are neurotoxic and we are now studying the underlying mechanisms involved in these effects. We will now develop therapeutic strategies for preventing the activation of these genes. Aim 4: Study of undiagnosed neuroinflammatory diseases. Many patients with neuroinflammatory diseases go undiagnosed for years until proper treatment. We have developed a protocol to study these patients to determine the underlying pathophysiology and initiate appropriate therapy. No data shoved different type of autoimmune and CNS infections have been studied via this mechanism.